Download Interpretation of Laboratory Tests: A Case

Interpretation of Laboratory Tests:
A Case-Oriented Review of Clinical
Laboratory Diagnosis
Roger L. Bertholf, Ph.D.
Associate Professor of Pathology
University of Florida Health Science Center/Jacksonville
1
Case 1: Oliguria and hematuria
2
Case 1: Oliguria and hematuria
A 7-year-old boy was brought to the pediatrician because of vomiting
and malaise. On physical examination he was slightly flushed, and had
some noticeable swelling of his hands and feet. The patient was
uncomfortable, and complained of pain “in his tummy”. He had a slight
fever. Heart was normal and lungs were clear. His past medical history
did not include any chronic diseases. The mother noted that he had a
severe sore throat “about two weeks ago”, but that it had cleared up on
its own. The child was not taking any medications. There were no
masses in the abdomen, and lymphadenopathy was not present. The
child had some difficulty producing a urine specimen, but finally was
able to produce a small amount of urine, which was dipstick-positive
for blood and protein.
R. Bertholf
American Society of Clinical Pathologists
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Questions. . .
• What is the differential diagnosis in this case?
• What laboratory tests might be helpful in
establishing the diagnosis?
R. Bertholf
American Society of Clinical Pathologists
4
What do the kidneys do?
•
•
•
•
•
Regulate body fluid osmolality and volume
Regulate electrolyte balance
Regulate acid-base balance
Excrete metabolic products and foreign substances
Produce and excrete hormones
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American Society of Clinical Pathologists
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The kidneys as regulatory organs
“The kidney presents in the highest degree the phenomenon
of sensibility, the power of reacting to various stimuli in a
direction which is appropriate for the survival of the organism;
a power of adaptation which almost gives one the idea that its
component parts must be endowed with intelligence.”
E. Starling (1909)
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Review of Renal Anatomy and Physiology
• The kidneys are a pair of fist-sized organs that are
located on either side of the spinal column just
behind the lower abdomen (L1-3).
• A kidney consists of an outer layer (renal cortex)
and an inner region (renal medulla).
• The functional unit of the kidney is the nephron;
each kidney has approximately 106 nephrons.
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Renal anatomy
Cortex
Pelvis
Capsule
Medulla
To the bladder
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The Nephron
Proximal tubule
Afferent arteriole
Distal tubule
Glomerulus
Bowman’s capsule
Collecting duct
Renal artery
Henle’s Loop
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Glomerular filtration
Vascular space
Glomerlular
capillary
membrane
Bowman’s space
Mean capillary blood
pressure = 50 mm Hg
 2,000 Liters
per day
 200 Liters
per day
BC pressure = 10 mm Hg
(25% of cardiac output)
Onc. pressure = 30 mm Hg
Net hydrostatic = 10 mm Hg
R. Bertholf
GFR  130 mL/min
American Society of Clinical Pathologists
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What gets filtered in the glomerulus?
• Freely filtered • Some filtered
• None filtered
– H2O
– Immunoglobulins
– 2-microglobulin
– Na+, K+, Cl-,
– RBP
– Ferritin
HCO3-, Ca++,
– Cells
– 1-microglobulin
Mg+, PO4, etc.
– Albumin
– Glucose
– Urea
– Creatinine
– Insulin
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Then what happens?
• If 200 liters of filtrate enter the nephrons each day,
but only 1-2 liters of urine result, then obviously
most of the filtrate (99+ %) is reabsorbed.
• Reabsorption can be active or passive, and occurs
in virtually all segments of the nephron.
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Reabsorption from glomerular filtrate
% Reabsorbed
Water
Sodium
Potassium
Chloride
Bicarbonate
Glucose
Albumin
Urea
Creatinine
R. Bertholf
99.2
99.6
92.9
99.5
99.9
100
95-99
50-60
0 (or negative)
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How does water get reabsorbed?
• Reabsorption of water is passive, in response to
osmotic gradients and renal tubular permeability.
– The osmotic gradient is generated primarily by
active sodium transport
– The permeability of renal tubules is under the
control of the renin-angiotensin-aldosterone
system.
• The driving force for water reabsorption, the
osmotic gradient, is generated by the Loop of
Henle.
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The Loop of Henle
Proximal tubule
Distal tubule
Na+
Ascending loop
H 2O
Descending loop
Increasing osmolality
Renal Cortex
300 mOsm/Kg
Na+
Na+
Na+
Renal Medulla
1200 mOsm/Kg
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Regulation of distal tubule Na+
permeability
JGA
 Na+
 BP
Renin
Angiotensinogen
Angiotensin I
Angiotensin II
vasoconstriction
Angiotensin III
Aldosterone
Adrenal cortex
Na+
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Na+
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Regulation of H2O reabsorption
Pituitary
Plasma
hyperosmolality
ADH (vasopressin)
H2O
H2O
Renal Medulla (osmolality 1200 mOsm/Kg)
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American Society of Clinical Pathologists
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Summary of renal physiology
TRPF (Filtered and secreted)
Filtration - Reabsorption + Secretion = Elimination
GFR (Filtered but not reabsorbed or secreted)
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Measurement of GFR
 Cu  Vu ( 24h ) 
Clearance  
  0.694
 C p

Cu = Concentration in urine
Vu(24h) = 24-hour urine volume
Cp = Concentration in plasma
0.694 = 1000 mL/1440 min
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American Society of Clinical Pathologists
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Compounds used to measure GFR
• Should not be metabolized, or alter GFR
• Should be freely filtered in the glomeruli, but neither
reabsorbed nor secreted
• Inulin (a polysaccharide) is ideal
• Creatinine is most popular
– There is some exchange of creatinine in the tubules
– As a result, creatinine clearance overestimates GFR by
about 10% (But. . .)
• Urea can be used, but about 40% is (passively)
reabsorbed
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American Society of Clinical Pathologists
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Relationship between creatinine and GFR
Plasma creatinine
6
5
4
3
2
1
0
0
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20
40
60
80
100
GFR (mL/min)
American Society of Clinical Pathologists
120
140
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Measurement of TRPF
• Para-aminohippurate (PAH) is freely filtered in the
glomeruli and actively secreted in the tubules.
• PAH clearance gives an estimate of the total
amount of plasma from which a constituent can be
removed.
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American Society of Clinical Pathologists
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Creatinine
Creatine
Creatinine
1-2% of creatine is hydrolyzed to creatinine each day
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Jaffe method for creatinine
Janovsky Complex
max = 490-500 nm
Max Eduard Jaffe (1841-1911), German physiologic chemist
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Modifications of the Jaffe method
• Fuller’s Earth (aluminum silicate, Lloyd’s reagent)
– adsorbs creatinine to eliminate protein interference
• Acid blanking
– after color development; dissociates Janovsky
complex
• Pre-oxidation
– addition of ferricyanide oxidizes bilirubin
• Kinetic methods
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0
R. Bertholf
A
t
Slow-reacting
(protein)
A
 rate
t
Fast-reacting
(pyruvate, glucose, ascorbate)
Absorbance ( = 520 nm)
Kinetic Jaffe method
creatinine (and -keto acids)
20
Time (sec) 
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80
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Enzymatic creatinine methods
• Creatininase
– creatininecreatineCKADPPKLD
• Creatinase
– creatininecreatinesarcosinesarcosine
oxidaseperoxideperoxidase reaction
• Creatinine deaminase (iminohydrolase)
– most common
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Creatinine deaminase method
Creatinine
Sarcosine
Creatinine
iminohydrolase
+ H2O
NCS
amidohydrolase
- NH3, CO2
Sarcosine oxidase
N-Methylhydantoin
ATP
NMH amidohydrolase
N-Carbamoylsarcosine
ADP
Formaldehyde + glycine
+ O2
H2O
H2O2
Oxygen receptor
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H2O
Peroxidase
Colored product
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Measurement of urine protein
• Specimen
– Timed 24-h is best
– Urine protein/creatinine ratio can be used with
random specimen
• Normal protein excretion is <150 mg/24h
– 50-60% albumin
– Smaller proteins (1-, 2-microglobulins)
– Tamm-Horsfall (uromucoid, secreted by tubules)
– IgA, tubular epithelial enzymes, and other nonfiltered components
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American Society of Clinical Pathologists
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Dipstick method for urine protein
• Method is based on protein association with pH
indicator
• Test pad contains dye tetrabromphenol blue at
pH=3
• If protein binds to the pH indicator, H+ is displaced
and the color changes from yellow to green (or
blue)
• Most sensitive to albumin (poor method for
detecting tubular proteinuria)
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American Society of Clinical Pathologists
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What causes excess urinary protein?
• Overload proteinuria
– Bence-Jones (multiple myeloma)
– Myoglobin (crush injury, rhabdomyolysis)
– Hemoglobin
• Tubular proteinuria
– Mostly low MW proteins (not albumin)
– Fanconi’s, Wilson’s, pyelonephritis, cystinosis
• Glomerular proteinuria
– Mostly albumin at first, but larger proteins appear
as glomerular membrane selectivity is lost.
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American Society of Clinical Pathologists
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Classification of proteinuria: Minimal
•
•
•
•
•
<1 gram of protein per day
Chronic pyelonephritis
Mild glomerular disease
Nephrosclerosis (usually due to hypertension)
Chronic interstitial nephritis (usually analgesicrelated)
• Renal tubular disease
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American Society of Clinical Pathologists
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Classification of proteinuria: Moderate
•
•
•
•
1.0 - 4.0 grams of protein per day
Usually associated with glomerular disease
Overflow proteinuria from multiple myeloma
Toxic nephropathies
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American Society of Clinical Pathologists
33
Classification of proteinuria: Severe
• >4 grams of protein per day
• Nephrotic syndrome (GBM permeability)
– Sx: edema, proteinuria, hypoalbuminemia,
hyperlipidemia
– In adults, usually 2 to systemic disease (SLE,
diabetes)
– In children, cause is usually primary renal disease
• Minimal Change Disease (Lipoid Nephrosis)
– Most common cause of NS in children
– Relatively benign (cause unknown, not autoimmune)
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American Society of Clinical Pathologists
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Proteinuria due to glomerulonephritis
• Acute, rapidly progressive, or chronic GN can
result in severe proteinuria
• Often the result of immune reaction (Circulating
Immune-Complex Nephritis)
– Antigen can be endogenous (SLE) or exogeneous
– Glomerular damage is mostly complementmediated
– If antigen is continuously presented, GN can
become chronic
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American Society of Clinical Pathologists
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How do red blood cells get in urine?
• Hematuria can result from bleeding anywhere in
the kidneys or urinary tract
– Disease, trauma, toxicity
• Hemoglobinuria can result from intravascular
hemolysis
– Disease, trauma, toxicity
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American Society of Clinical Pathologists
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Dipstick method for hemoglobin
Heme
H2O2 + chromogen*
Peroxidase
Oxidized chromogen + H2O
• Ascorbic acid inhibits the reaction, causing a false
negative test
• Depends on RBC lysis (may not occur in urine
with high specific gravity)
• Detection limit approximately 10 RBC/L
*tetramethylbenzidine; oxidized form is green
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American Society of Clinical Pathologists
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Microscopic examination of urine sediment
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American Society of Clinical Pathologists
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Significance of RBC casts in urine
•
•
•
•
Indicative of blood crossing the GBM
Casts form in the distal tubules
Stasis produces brown, granular casts
RBC casts almost always reflect glomerular disease
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American Society of Clinical Pathologists
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Bright’s Disease (acute glomerulonephritis)
• Characterized by oliguria, proteinuria, and
hematuria
• Most common cause is immune-related
Richard Bright (1789-1858)
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Primary Glomerulonephritis
• Proliferative GN
– Acute Post-infectious GN
– Idiopathic or Crescentic GN
– -GBM disease
– Membranoproliferative GN
• Focal GN
– IgA nephropathy
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American Society of Clinical Pathologists
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Primary Glomerulonephritis, cont.
• Idiopathic membranous GN
– Histological diagnosis, probably immune complex
• Chronic GN
– Clinical Dx; many potential causes
• Lipoid Nephrosis
– Histological findings normal; “Nephrosis”
• Focal Glomerular Sclerosis
– Probably immune (IgM) related
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American Society of Clinical Pathologists
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Secondary Glomerulonephritis
• Systemic Lupus Erythematosus
– Renal failure accounts for 50% of SLE deaths
• Polyarteritis (inflammatory vasculitis)
• Wegener’s Granulomatosis (lung and URT)
• Henoch-Schönlein Syndrome
– Lacks edema assoc. with post-streptococcal GN
• Goodpasture’s Syndrome (pulmonary hemorrhage)
• Hemolytic-Uremic Syndrome
• Progressive Systemic Sclerosis (blood vessels)
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American Society of Clinical Pathologists
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Case 3: Chest Pain
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Case 3: Chest Pain
A 63 year old male was brought to the emergency department
after complaining of severe chest pain that had lasted for two
hours. He had been mowing his lawn when the pain developed,
and he became concerned when the pain did not subside after
he stopped the activity. He had no previous history of heart
disease. On presentation he was moderately overweight, diaphoretic, and in obvious discomfort. He described his chest
pain as “beginning in the center of my chest, then my arms,
neck, and jaw began to ache too.”
Diagnostic procedures were performed.
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American Society of Clinical Pathologists
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Questions
• What is the most important consideration in the
triage of this patient?
• What tests should be ordered?
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American Society of Clinical Pathologists
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Chest pain
• One of the most common reasons for seeking
medical attention
• Characteristics of cardiogenic chest pain (angina)
– induced by exercise
– described as “pressure”
– radiates to extremities
– MI not relieved by rest or vasodilatory drugs (NG)
• Only 25% of patients presenting with chest pain as
the primary complaint will ultimately be diagnosed
as MI (specificity=25%; sensitivity=80%)
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The Heart
Aorta
Pulmonary arteries
Superior vena cava
LA
RA
LV
RV
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The Heart (posterior view)
Aorta
Superior vena cava
Pulmonary arteries
Pulmonary veins
Inferior vena cava
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Cardiac physiology
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Cardiac conduction system
Sinoatrial (SA) node
Atrioventral (AV) node
His bundle
Left bundle branch
Right bundle branch
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Normal Electrocardiogram
R
T
U
P
Q
S
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Myocardial infarction
Left coronary artery
Anterior left ventricle
Right coronary artery
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ECG changes in myocardial infarction
R
S-T elevation
T
P
Q
S
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Diagnostic value of ECG
• ECG changes depend on the location and severity of
myocardial necrosis
• Virtually 100% of patients with characteristic Qwave and S-T segment changes are diagnosed with
myocardial infarction (100% specificity)
• However, as many as 50% of myocardial infarctions
do not produce characteristic ECG changes
(sensitivity  50%)
• ECG may be insensitive for detecting prognostically
significant ischemia
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History of cardiac markers
• 1975: Galen describes the use of CK, LD, and
isoenzymes in the diagnosis of myocardial infarction.
• 1980: Automated methods for CK-MB (activity) and
LD-1 become available.
• 1985: CK-MB isoforms are introduced.
• 1989: Heterogeneous immunoassays for CK-MB
(mass) become available.
• 1991: Troponin T immunoassay is introduced.
• 1992: Troponin I immunoassay is introduced.
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American Society of Clinical Pathologists
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Enzyme markers
• Aspartate transaminase (AST; SGOT)
• 2-Hydroxybutyrate dehydrogenase
• Lactate dehydrogenase
– Five isoenzymes, composed of combinations of H
(heart) and M (muscle) subunits
• Creatine kinase
– Three isoenzymes, composed of combinations of
M (muscle) and B (brain) subunits
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Lactate dehydrogenase (LD)
NAD+
NADH
Pyruvate
LD
Lactate
• LD activity is measured by monitoring absorbance
at  = 340 nm (NADH)
• Methods can be P  L or L  P
– But. . .reference range is different
• Total LD activity has poor specificity
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Tissue specificity of LD isoenzymes
LD isoenzyme
Tissues
LD-1
Heart (60%), RBC, Kidney
LD-2
Heart (30%), RBC, Kidney
LD-3
Brain, Kidney
LD-4
Liver, Skeletal muscle, Brain, Kidney
LD-5
Liver, Skeletal muscle, Kidney
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LD isoenzyme electrophoresis (normal)
LD-2 > LD-1 > LD-3 > LD-4 > LD-5
LD-2
LD-1
LD-3
LD-4
LD-5
Cathode (-)
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Anode (+)
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LD isoenzyme electrophoresis (abnormal)
LD-1
LD-1 > LD-2
LD-2
LD-3
LD-4
LD-5
Cathode (-)
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Anode (+)
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Direct measurement of LD-1
• Electrophoresis is time-consuming and only semiquantitative
• Antibodies to the M subunit can be used to
precipitate LD-2, 3, 5, and 5, leaving only LD-1
– Method can be automated
– Normal LD-1/LDtotal ratio is less than 40%
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Sensitivity and specificity of LD-1
• Sensitivity and specificity of the LD 1:2 “flip”, or
LD-1 > 40% of total, are 90+% within 24 hours of
MI, but. . .
– May be normal for 12 or more hours after
symptoms appear (peak in 72-144 hours)
– May not detect minor infarctions
• Elevations persist for up to 10 days
• Even slight hemolysis can cause non-diagnostic
elevations in LD-1
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63
Creatine Kinase (CK)
Phosphocreatine
ADP
CK
Creatine
Mg++
ATP
Glucose
ADP
HK
Glucose-6-phosphate
NADP+
GPD
6-Phosphogluconate
NADPH
=340 nm
Oliver and Rosalki method (1967)
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64
Tissue specificities of CK isoenzymes
CK-1
(BB)
CK-2
(MB)
CK-3
(MM)
Skeletal muscle
0%
1%
99%
Cardiac muscle
1%
20%
79%
Brain
97%
3%
0%
Tissue
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Measurement of CK isoenzymes
• Electrophoresis (not used anymore)
• Immunoinhibition/precipitation
– Antibody to M subunit
– Multiply results by 2
– Interference from CK-1 (BB)
• Most modern methods use two-site (“sandwich”)
heterogeneous immunoassay
– Measures CK-MB mass, rather than activity
– Gives rise to a pseudo-percentage, often called the
“CK-MB index”
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Sensitivity/specificity of CK-MB
• Sensitivity and specificity of CK-MB for
myocardial infarction are >90% within 7-18 hours;
peak concentrations occur within 24 hours
• CK is a relatively small enzyme (MW = 86K), so it
is filtered and cleared by the kidneys; levels return
to normal after 2-3 days
• Sensitivity is poor when total CK is very high, and
specificity is poor when total CK is low
• Presence of macro-CK results in false elevations
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67
CK isoforms
C-terminal lysine
CK-MB2 (tissue)
Plasma carboxypeptidase
CK-MB1 (circulating)
• C-terminal lysine is removed from the M subunit-therefore, there are three isoforms of CK-3 (MM)
• t½: CK-MB1 > CK-MB2
• Ratio of CK-MB2 to CK-MB1 exceeds 1.5 within six
hours of the onset of symptoms
• Only method currently available is electrophoresis
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68
Myoglobin
• O2-binding cytosolic protein found in all muscle
tissue (functional and structural analog of
hemoglobin)
• Low molecular weight (17,800 daltons)
• Elevations detected within 1-4 hours after
symptoms; returns to normal after 12 hours
• Nonspecific but sensitive marker--primarily used
for negative predictive value
• Usually measured by sandwich, nephelometric,
turbidimetric, or fluorescence immunoassay
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Temporal changes in myoglobin and CK-MB
CK-MB
800
60
50
40
30
20
10
0
600
400
200
0
0
8
16
24
32
40
CK-MB (ug/L)
Myoglobin (ug/L)
Myoglobin
48
Time after symptoms
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70
Troponin
Tropomyosin
Actin
TnT (42 Kd)
TnI (23 Kd)
TnC
Myosin
Thick Filament
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Tissue specificity of Troponin subunits
• Troponin C is the same in all muscle tissue
• Troponins I and T have cardiac-specific forms,
cTnI and cTnT
• Circulating concentrations of cTnI and cTnT are
very low
• cTnI and cTnT remain elevated for several days
• Hence, Troponins would seem to have the
specificity of CK-MB (or better), and the long-term
sensitivity of LD-1
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72
Is cTnI more sensitive than CK/CK-MB?
79 y/o female with Hx of HTN, CHF, CRI, Type II diabetes
log X normal
1
0.5
CK
CK-MB
CK-MB Index
cTnI
0
-0.5
-1
1
8
40
66
Hours since presentation
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Measurement of cTnI and cTnT
• All methods are immunochemical (ELISA, MEIA,
CIA, ECIA)
• Roche Diagnostics (formerly BMC) is the sole
manufacturer of cTnT assays
– First generation assay may have had some crossreactivity with skeletal muscle TnT
– Second generation assay is cTnT-specific
– Also available in qualitative POC method
• Many diagnostics companies have cTnI methods
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74
W.H.O. has a Myocardial Infarction?
A patient presenting with any two of the following:
• A clinical history of ischemic-type chest discomfort
• Changes on serially obtained ECG tracings
• A rise and fall in serum cardiac markers
Source JACC 28;1996:1328-428
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Sensitivity/Specificity of WHO Criteria
100%
80%
60%
Sensitivity
Specificity
40%
20%
0%
Chest Pain ECG changes
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Serum
markers
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# of labs reporting
What Cardiac Markers do Labs Offer?
3500
3000
2500
2000
1500
1000
500
0
1997
1998
CK-MB
(ng/mL)
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CK-MB
(IU/L)
cTnI
cTnT
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